Biological methods for the production of chemicals have shown promise as alternatives to traditional chemical syntheses, particularly for complex biochemicals such as polypeptides and proteins. The full potential of these biological methods, which rely on self-reproducing life forms as chemical producers, has not yet been realized, however. Although several biological systems have been successfully explored as potential sources of a variety of chemicals, each system has been found to have its limitations.
The simplest and most thoroughly investigated biological methods for chemical production are microbiological systems. Primitive prokaryotic cells have been amenable to investigation and have been found to produce a variety of small organic and inorganic compounds, as well as a variety of complex biomolecules, such as homologous and heterologous polypeptides and proteins. The most extensively characterized prokaryote, Escherichia coli, synthesizes complex biomolecules using a relatively straightforward process of gene expression requiring minimal expression control elements and an uninterrupted coding region. Further, genetic elements encoding heterologous polypeptides can be introduced and expressed in E. coli without much difficulty. With these advantages, a wide variety of polypeptides have been expressed in a controlled manner in this organism. However, E. coli cultures do require the costly inputs of energy and nutrients. The organism also does not readily secrete the produced polypeptides, adding to the time and expense required to isolate the desired compound. Although other microbes, e.g., species of Bacillus, do secrete polypeptides into growth media, cultures of these organisms also require costly inputs of energy and nutrients. Moreover, all of these primitive prokaryotic systems exhibit additional shortcomings such as the expensive effort to avoid culture contamination and the inability of the microbes to properly process or derivatize many expressed polypeptides to fully biologically active forms.
Yeast, a fairly primitive eukaryotic cell, has also been used to produce chemicals, including heterologous polypeptides. Although these cells may do a better job of reproducing the natural derivatization of most commercially desirable (i.e., eukaryotic) polypeptides, the reproduction is imperfect. Additionally, cultures of yeast cells are also susceptible to contamination and the cells themselves require valuable resources in the forms of energy and nutrients, Efforts to obtain the desired chemicals, such as heterologous polypeptides, are also burdened by the frequent need to extract the chemical from the cell and purify that compound from the chemically complex contents of the yeast cell released during extraction.
Plant cells also reportedly produce chemicals such as native and heterologous polypeptides. Existing genetic transformation technologies allow the transfer of genes into a wide variety of plant cell lines. In addition, a number of regulatory elements and signal sequences have been found that facilitate the expression of heterologous gene products in plant cells. Further, plant cell cultures have been shown to secrete low levels of heterologous proteins. Pen et al., Bio/Technology 10:292-296 (1992) reported that a bacterial signal sequence for alpha-amylase would direct secretion of this protein from tobacco cells. Thus, the plant cell wall has been shown to be permeable to polypeptides as large as 150 kDa. These tools have been used to reportedly engineer plant cells to produce and secrete a variety of heterologous polypeptides, as noted by Esaka et al., Phytochem. 28:2655-2658 (1989), Esaka et al., Physiologia Plantarum 92:90-96 (1994), Esaka et al., Plant Cell Physiol. 36:441-446 (1995), and Li et al., Plant Physiol. 114:1103-1111 (1997). Other polypeptides produced in plant cells include the Hepatitis B surface antigen (HBsAg), the enterotoxin B (LT-B) subunit from E. coli (a diarrhea inducer), a variety of antibodies, human growth factors, and hormones. Although these cells secrete the desired polypeptides, these systems also require energy and nutrient inputs that add to the costs of polypeptide recovery.
Mammalian cells, although expected to closely approximate the native derivation of many important polypeptides (e.g., human polypeptides), are very costly to culture, due to their sensitivity to contaminants, their requirements for energy, gases, and nutrients, and their limited lifespans. Isolation of the produced chemicals also would be relatively expensive in view of the typical inability of mammalian cells to secrete products and the relative chemical complexity of the intracellular environment of these cells.
More complex life forms, such as tissues, have not been shown to be practical sources of chemicals such as heterologous polypeptides. In brief, tissue culture suffers from the disadvantages noted above in the context of discussing cell culture. Similarly, organ culture has not been a focus of chemical production on a commercial scale. In general, the cells, tissues, and organs of a multi-cellular organism require costly intervention to ensure their survival and productivity.
Organisms have also been recruited to produce desirable chemicals, including heterologous polypeptides expressed from transgenes. Organism-based methods are freed from some of the restrictions limiting the aforementioned methods. For example, a relatively limited set of chemical inputs can be converted by the synthetic capacity of an organism into the full set of chemicals required to live and grow. Perhaps for this reason, organism-based methods appear to be hardier than cell-, tissue-, or organ-based methods for chemical production. Nevertheless, organisms, particularly heterotrophic organisms such as animals, do require chemical inputs to provide sources of energy and essential nutrients. Frequently, efforts must also be made to ward off disease resulting from, e.g., infection.
Plants, as photoautotrophic organisms, provide an alternative to heterotrophic animals as life forms for the production of chemicals. Transgenic plants have been generated, albeit typically to improve the characteristics of the plants themselves (e.g., to confer resistance to disease, to improve the yield of edible foodstuffs). Nevertheless, some transgenic plants have been used to produce chemicals such as heterologous polypeptides. As noted above, a variety of expression control sequences (e.g., regulatory elements, signal peptide sequences) have been found to function in plant cells generally, or to preferentially function in the cells of particular plant tissues and organs. For example, Sijimons et al. (U.S. Pat. No. 5,650,307) expressed Human Serum Albumin (HSA) by fusing the HSA coding region to the leader sequence from Alfalfa Mosaic Virus. This fused coding region was placed under the control of the Cauliflower Mosaic Virus 35S promoter and the Nopaline Synthase terminator. The HSA was expressed in transgenic potato plants and transgenic tobacco cells. Sijmons et al. further disclosed the secretion of HSA by potato plant cells and recovery of the heterologous HSA from the intercellular space of those plants. Of course, this recovery method involved the destruction of the potato plants.
U.S. Pat. No. 5,580,768 also discloses the production and secretion of heterologous protein by a plant. In particular, the '768 Patent discloses a transgenic rubber tree, with the expressed transgene protein being collected from wounds as a part of the latex. This system is highly specialized for use with Hevaea species (perennial tree species with slow growth), and the tree must be damaged by wounding to recover the heterologous polypeptide in the form of a latex mixture.
Transformed plant material has also been used to express heterologous polypeptides. Wongsamuth et al., Biotech. and Bioengineer. 54:401-415 (1997), report the use of hairy root cultures to express murine IgG.sub.1 monoclonal antibody. Further, some antibody activity was found in the medium of the hairy root cultures maintained under axenic conditions as heterotrophic biomasses requiring costly energy and nutrient inputs.
Thus, a need continues to exist in the art for biological methods for economically producing and recovering a wide variety of chemicals, including heterologous polypeptides, in quantity and without destruction of the biological producer.